Qualitative Analysis of Lipids

Prepared by:
KATYA AMELIA A. VALIDO, LPT
 Lipids
• Refers to both true and fat-like substances. These are biomolecules
that are insoluble in water but are soluble in organic solvents. The
water insolubility is due to the fact that the polar portion of their
molecule is much smaller than the non-polar portion.
• Important function of lipids is the storage of energy in the form of
body fat.
• Fatty acids, one of the building blocks of lipids, is just one of the
examples of the hydrolysis products of fats and oils as well as of the
compound lipids.
 Qualitative Analysis of Lipids
• Helps us determine the presence or absence of lipid, depending upon
the color change.
• The qualitative analysis of lipid is an analytical method that detects
lipids by the characteristic change in sample’s color.
• Spotting Effect
• Solubility
• Test for Unsaturation (Bromine Water Test)
• Acrolein Test
• Extraction of Brain Lipids
• Detection of Brain Lipids
-Ninhydrin Test
-Ammonium Molybdate Test
-Leibermann-Burchard Test
Spotting Effect
 Spotting Effect
• A preliminary test for the lipids, which is characterized by a
translucent and greasy spot.
 What is the principle of the test?
• The lipid will not wet the filter paper, unlike water.
• The lipids will form a greasy or translucent spot due to their greasy
texture, and penetrate the filter paper. Unlike lipids, the spot of water
will disappear from the paper.
 What is the purpose of the test?
• Confirms the presence of fats in the given sample.
 Method
1. Take a filter paper.
2. Add one drop of water at one end and a drop of oil or lipid at the
other end.
3. Observe the appearance of a translucent spot on the filter paper.
 Result

Biology Reader
• Positive result: Translucent spot will appear on the filter paper.
• Negative result: Translucent spot will not appear on the filter paper.
 WITH OR WITHOUT
SAMPLES
TRANSLUCENT SPOT

Vegetable oil With translucent spot

Hair wax With minimal translucent spot

Mayonnaise With translucent spot

Lotion With minimal translucent spot

Lecithin With translucent spot

Why would a lipid leave a translucent spot on
paper?
• * Like water, the lipids soak into the paper fibers, but the lipid spot
evaporates much slowly than water (because the forces that hold the
lipid molecules together are stronger than those in water molecules).
So the spot remains.
 What property of lipids is detected by this
test?
• Lipids are oily or greasy nonpolar molecules
Solubility Test
 Solubility Test
• It is the preliminary test that detects the presence of all lipids.
• Solubility test detects lipid solubility in various solvents to check
whether it is miscible of immiscible in polar or non-polar solvents.
• Lipids are readily miscible in non-polar solvents

• *Miscibility is the property of two substances to

completely mix to form a homogenous solution.
• * Immiscibility is the property of two substances
that do not completely mix to form solution.
• Polar Solvents: are the solvents that contain partial negative and
partial positive charge or we can say they must have a dipole
moment.
• Polar solvents generally dissolve other polar substances.
• Examples: water, acetone, methanol, etc.
• Non-polar solvents: are solvents that do not have dipole moment or
do not contain any partial positive and partial negative charge.
• *Non-polar solvents do not dissolve in polar solvents as they do not
carry any charge.
• Non-polar solvents are dissolved in non-polar compounds.
• Examples: benzene, hexane, toluene
 Method
1. Prepare clean and dry test tubes. (Assume that your solvents are;
water, methylene chloride, ether, and toluene)
2. Place 1.0mL of these solvents into their respective containers.
3. In each test tube, add 3 drops of vegetable oil (sample) and mix
thoroughly.
4. Compare the solubility of vegetable oil in the different solvents.
Result
• Positive result: lipids are
soluble in a non-polar
solvent
• Negative Result: lipids are
insoluble in a polar
solvent
• *lipids are non-polar
Solubility of vegetable oil in different solvents:
A. Water
• Fats and oils are nonpolar, so they will remain separate from
molecules of a polar solvent such as water.
• Oil and water do not mix because water molecules are more attracted
to each other than to oil molecules.
B. Methylene Chloride
• Methylene chloride is polar, but not as much polar as water because
of its nonpolar C-H bond, thus it will still mix with vegetable oil, but
not completely.
C. Ether
• Since ether and vegetable oil are both non-polar and insoluble in
water, they will mix together.

A class of organic compounds

that are used in perfumes, dyes,
oils, waxes, and other industrial
uses.
D. Toluene
• Toluene is composed of C-H bonds making it a non-polar, so
vegetable oil has the ability to dissolve in it.
Solubility of lecithin in different solvents:
A. Water
• The structure of lecithin is very similar with vegetable oil, but instead
of the third fatty acid, it has a phosphate group, which is polar thus it
is water soluble, which means it will slightly dissolve in water.
Phospholipids, like lecithin, emulsify oil in water by forming little
protective envelopes around tiny droplets of oil.
B. Methylene Chloride
• It is also a non-polar substance because of its C-H bond and most of
the lecithin dissolved in it even though it has polar ends.
C. Ether
• Lecithin attracts both water and fatty substances
• Ether is a non-polar substance and most of the lecithin dissolved it
even though it has polar ends
D. Toluene
• Also known as, methylbenzene, also consists of hydrocarbon chain
making it a non-polar substance.
• Most of the lecithin dissolved in it even though it has polar ends.
Therefore, the rule “like dissolves like” does apply to them.
 Solvents
Samples Methylene
Water chloride Ether Toluene
(polar) ***(polar) (non-polar) (non-polar)

Vegetable oil
(non-polar) Not mixed Mixed Mixed Mixed

Lecithin
***(polar and
non-polar) Slightly mixed Mixed Mixed Mixed
***emulsifier
Result
• If it separated into two layers, the oil is not dissolved. But if one layer
homogenous transparent formed, oil is dissolved in the solvent.
Test for Unsaturation: Bromine Water Test
 Test for Unsaturation: Bromine Water Test
• It is a qualitative test used to detect the unsaturated fatty acids or
double bond in a lipid sample.
What is the principle involved in the bromine
water test?
• Double bonds are found in the structure of unsaturated fatty acids
• If no change in color---it is saturated lipid
• If it becomes colorless--- it is unsaturated lipid
Acrolein Test
 Acrolein Test
• Acrolein test is used to detect the presence of glycerol or fat.
• Glycerol- a trihydroxy alcohol with three carbons atoms and three
hydroxyl groups
- basic building block of lipids
 What is the principle involved in Acrolein
Test?
• When fat is treated strongly in the presence of a dehydrating agent
like potassium bisulphate (KHSO4), the glycerol portion of the
molecule is dehydrated to form an unsaturated aldehyde, acrolein
that has a pungent irritating odour.
 Sample Odor of the Vapor

Glycerol Acrolein (pungent odor)

Olive oil Acrolein (burnt odor)

Acrolein (burnt odor)

Lecithin

• If there is a pungent irritating odor, then the presence of fats or oil is confirmed.
Detection of Brain Lipids
 Ninhydrin Test
• It is a chemical test used to the presence of amino acids, peptides,
and proteins
 What is the principle involved in the
Ninhydrin Test?
• This test involves the addition of ninhydrin reagent to the test sample
that results in the formation of deep blue color, often termed as
Ruhemann’s purple.
Result
Positive test: Blue-purple
and yellow reaction
products positively
identify free amino
groups on amino acids
and proteins.

Negative test: No change

(absence of amino acids
and proteins)
Ammonium Molybdate Test
 Ammonium Molybdate Test
• Test for inorganic phosphate
• Hydrolysis of pyrophosphate to phosphate forming yellow ppt

• When a mixture containing phosphate is heated with conc. HNO3 and

Ammonium Molybdate, yellow ppt of Ammonium Molybdate is
formed.
 Liebermann-Burchard Test
• To detect the presence of cholesterol.
• Cholesterol reacts with the strong concentrated acid, i.e. sulphuric
acid and acetic anhydride. Sulphuric acid and acetic anhydride act as
a dehydrating and oxidizing agent.
• Positive result: It indicates cholesterol in a sample by giving
bluish-green colour to the solution.
• Negative result: The colour of the solution will not change.